RESUMO
Between May 2006 and March 2007, 65 water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, the capital of the Brazilian state of Rio Grande do Sul. The aim was to explore the problem posed by, and the pathogenic potential of, Acanthamoeba in the pools. Free-living amoebae in the samples were isolated by culture with Escherichia coli and identified from trophozoite and cyst morphology and the results of a PCR with Acanthamoeba-specific oligonucleotide primers. Potential pathogenicity was assessed in osmotolerance and thermotolerance assays. Thirteen (20%) of the water samples investigated were found positive for free-living amoebae, all identified as belonging to morphological groups II (nine isolates) or III (four isolates) of the genus Acanthamoeba. All 13 isolates were found positive in the Acanthamoeba-specific PCR, and the results of the tolerance assays indicated that five (38%) of the isolates should be considered potentially pathogenic. The results of this first study on the occurrence and distribution of Acanthamoeba in the water of swimming pools in Porto Alegre confirm the presence of potentially pathogenic types that may present a risk to human health.
Assuntos
Acanthamoeba/isolamento & purificação , Piscinas , Acanthamoeba/genética , Acanthamoeba/patogenicidade , Animais , Brasil , Temperatura AltaRESUMO
Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.
Assuntos
Antígenos de Helmintos/isolamento & purificação , Equinococose/imunologia , Echinococcus/imunologia , Animais , Antígenos de Helmintos/imunologia , Área Sob a Curva , Estudos de Casos e Controles , Equinococose/diagnóstico , Epitopos/imunologia , Epitopos/isolamento & purificação , Escherichia coli/imunologia , Humanos , Imunoglobulina G/análise , Curva ROC , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Testes SorológicosRESUMO
Two different Echinococcus granulosus antigen B subunits (AgB8/1 and AgB8/2) were characterized and the structure of the genes encoding these two proteins were compared. DNA sequences were expressed in Escherichia coli and the antigens' diagnostic value was then assessed. The genomic sequence of AgB8/1 has a 92 bp intron in the position corresponding to amino acid 16; the AgB8/2 genomic sequence presents a 68 bp intron in the position corresponding to amino acid 20. Both introns are located between the putative N-terminal hydrophobic sequence and the secreted peptide. A comparison between the AgB8/1 and AgB8/2 nucleotide sequences showed a 53.5% identity among exons and a 50% identity between introns. According to the molecular diversity analysis, the elapsed time since both genes shared a common ancestor would be around 4.2x10(7) years. When the native AgB and the two recombinant antigens (rAgB8/1 and rAgB8/2) were tested in an anti-IgG ELISA, the sensitivity of the native antigen B was 77.41% and its specificity was 81.9%, while rAgB8/1 showed 54.84% of sensitivity and 80.17% of specificity and rAg138/2 had an 83.87% sensitivity and a 98.28% specificity. Statistical analysis confirms that rAgB8/2 has a better performance than rAgB8/1 and native AgB in ELISA.